1. Field of the Invention
This invention relates to the detection of small magnetized particles (beads) by a GMR sensor, particularly when such particles or beads are attached to molecules whose presence or absence is to be determined in a chemical or biological assay.
2. Description of the Related Art
GMR (giant magnetoresistive) devices have been proposed as effective sensors to detect the presence of specific chemical and biological molecules (the “target molecules”) when, for example, such target molecules are a part of a fluid mixture that includes other molecules whose detection is not necessarily of interest. The basic method underlying such magnetic detection of molecules first requires the attachment of small magnetic (or magnetizable) particles (also denoted “beads”) to all the molecules in the mixture that contains the target molecules. The magnetic beads are made to attach to the molecules by coating the beads with a chemical or biological species that binds to the molecules in the mixture. Then, a surface (i.e., a solid substrate) is provided on which there has been affixed receptor sites (specific molecules) to which only the target molecules will bond. After the mixture has been in contact with the surface so that the target molecules have bonded, the surface can be flushed in some manner to remove all unbonded molecules. Because the bonded target molecules (as well as others that have been flushed away) are equipped with the attached magnetic beads, it is only necessary to detect the magnetic beads to be able, at the same time, to assess the number of captured target molecules. Thus, the magnetic beads are simply “flags,” which can be easily detected (and counted) once the target molecules have been captured by chemical bonding to the receptor sites on the surface. The issue, then, is to provide an effective method of detecting the small magnetic beads, since the detection of the beads is tantamount to detection of the target molecules.
One prior art method of detecting small magnetic beads affixed to molecules bonded to receptor sites is to position a GMR device beneath them; for example, to position it beneath the substrate surface on which the receptor sites have been placed.
FIG. 1 is a highly schematic diagram (typical of the prior art methodology) showing a magnetic bead (10) covered with receptor sites (20) that are specific to bonding with a target molecule (30) (shown shaded) which has already bonded to one of the sites. A substrate (40) is covered with receptor sites (50) that are also specific to target molecule (30) and those sites may, in general, be different from the sites that bond the magnetic particle to the molecule. The target molecule (30) is shown bonded to one of the receptor sites (50) on the surface.
A prior art GMR sensor (60), shown without any detail, is positioned beneath the receptor site. As shown schematically in the cross-sectional view of FIG. 2a, the prior art GMR sensor ((60) in FIG. 1) (60), is preferably in the form of a laminated thin film stripe that includes a magnetically free layer (610) and a magnetically pinned layer (620) separated by a thin, non-magnetic but electrically conducting layer (530). Typically, the sensor will also include a capping layer or overlayer (550). The GMR properties of such a film stripe causes it to act essentially as a resistor whose resistance depends on the relative orientation of the magnetic moments of the free and pinned layers (shown here as arrows (640), (650) both directed out of the figure plane). FIG. 2b shows, schematically, an overhead view of the sensor of FIG. 2a, showing more clearly the direction of the magnetic moments (640) and (650), shown dashed, as it is below (640). Also shown are two lobes outlining a region of equal strength of an external magnetic field B (800). The field strength is shown directionally as arrows (160) that would be produced by a magnetized particle (not shown here) that is positioned above the sensor, as shown in FIG. 1. This will also be shown more clearly in FIG. 3, below. The field of the lobes will deflect (640) (deflection not shown), but leave (650) unchanged.
FIG. 3 shows, schematically, a magnetic particle (10) situated (by binding) over a surface layer (45) formed on a substrate (40) in a typical prior art configuration. The surface layer is required to support the bonding sites and can be a layer of Si3N4 and the substrate can be a Si substrate on or within which the required circuitry can be formed. For simplicity, a target molecule is not shown. A GMR sensor (60), as illustrated in any of the previous figures, is positioned between the surface layer and the substrate (40). An electromagnet (100) is positioned beneath the substrate and creates a magnetic field H (120) directed vertically through the substrate, the GMR sensor, and the magnetic bead. The external field, H, is directed perpendicularly to the magnetic moments of the GMR sensor (640), (650) so as not to change their relative orientations. Because of the magnetic properties of the bead, the external field H (120), induces a magnetic moment M (150), shown as an arrow in the bead which, in turn, produces a magnetic field B (160) that extends beyond the boundary of the bead as shown by the dashed lobes. The magnetic field B (160), in turn, penetrates the plane of the sensor and its component in that plane (shown as the lobes (800) in FIG. 2b) can change the orientation of the magnetic moment of the sensor free layer as is shown schematically in FIGS. 4a and 4b. 
The magnetization of the free layer (640), is now changed in direction relative to the magnetization of the pinned layer (650), because of the presence of the magnetic field of the magnetized bead (160) that is directed within the plane of the free layer. Because the presence of the magnetized bead affects the magnetic moment of the free layer, it thereby, changes the resistance of the GMR sensor strip. By detecting the changes in resistance, the presence or absence of a magnetized bead is made known and, consequently, the binding of a target molecule is detected. Ultimately, an array of sensors can be formed beneath a substrate of large area that is covered by a large number of binding sites. The variation of the resistance of the sensor array is then a good indication of the number of target molecules that has been captured at sites and that number, in turn, can be related to the density of such target molecules in the mixture being assayed.
As is well known by those skilled in the field, although the magnetization of the free layer moves in response to external magnetic stimuli during operation of the sensor, the magnetization of the free layer is preferably fixed when the sensor is in a quiescent mode and not acted on by external fields. The fixing of the free layer magnetization under these conditions is called “biasing” the free layer and the position of the magnetic moment of the free layer in this position is called its bias point. It is also known to those skilled in the art that the bias position of the free layer is subject to the effects of hysteresis, which means that the bias position is not maintained after the magnetization of the free layer is made to cycle through positive and negative directions by external magnetic stimuli and a quiescent state is once again achieved. This hysteresis has a negative impact on the reproducibility of sensor readings, particularly when the external stimuli moving the free layer magnetization are small to begin with. One of the objects of the present invention will be to eliminate the adverse effects of hysteresis. Given the increasing interest in the identification of biological molecules it is to be expected that there is a significant amount of prior art directed at the use of GMR sensors (and other magnetic sensors) to provide this identification. A detailed research paper that presents an overview of several different approaches as well as the use of GMR sensors is: “Design and performance of GMR sensors for the detection of magnetic microbeads in biosensors” J. C. Rife et al., Sensors and Actuators A 107 (2003) 209-218. An early disclosure of the use of magnetic labels to detect target molecules is to be found in Baselt (U.S. Pat. No. 5,981,297). Baselt describes a system for binding target molecules to recognition agents that are themselves covalently bound to the surface of a magnetic field sensor. The target molecules, as well as non-target molecules, are covalently bound to magnetizable particles. The magnetizable particles are preferably superparamagnetic iron-oxide impregnated polymer beads and the sensor is a magnetoresistive material. The detector can indicate the presence or absence of a target molecule while molecules that do not bind to the recognition agents (non-target molecules) are removed from the system by the application of a magnetic field.
A particularly detailed discussion of the detection scheme of the method is provided by Tondra (U.S. Pat. No. 6,875,621). Tondra teaches a ferromagnetic thin-film based GMR magnetic field sensor for detecting the presence of selected molecular species. Tondra also teaches methods for enhancing the sensitivity of GMR sensor arrays that include the use of bridge circuits and series connections of multiple sensor stripes. Tondra teaches the use of paramagnetic beads that have very little intrinsic magnetic field and are magnetized by an external source after the target molecules have been captured.
Coehoorn et al. (US Pub. Pat. Appl. 2005/0087000) teaches a system that is similar to that of Tondra (above), in which magnetic nanoparticles are bound to target molecules and wherein the width and length dimensions of the magnetic sensor elements are a factor of 100 or more larger than the magnetic nanoparticles.
Prinz et al. (U.S. Pat. No. 6,844,202) teaches the use of a magnetic sensing element in which a planar layer of electrically conducting ferromagnetic material has an initial state in which the material has a circular magnetic moment. In other respects, the sensor of Prinz fulfills the basic steps of binding at its surface with target molecules that are part of a fluid test medium. Unlike the GMR devices disclosed by Tondra and Coehoorn above, the sensor of Prinz changes its magnetic moment from circular to radial under the influence of the fringing fields produced by the magnetized particles on the bound target molecules.
Gambino et al. (U.S. Pat. No. 6,775,109) teaches a magnetic field sensor that incorporates a plurality of magnetic stripes spaced apart on the surface of a substrate in a configuartion wherein the stray magnetic fields at the ends of the stripes are magnetostatically coupled and the stripes are magnetized in alternating directions.
Simmonds et al. (U.S. Pat. No. 6,437,563) teaches a method of detecting magnetic particles by causing the magnetic fields of the particles to oscillate and then detecting the presence of the oscillating fields by inductively coupling them to coils. Thus, the sensor is not a GMR sensor as described above, but, nevertheless, is able to detect the presence of small magnetic particles.
Finally, Sager et al. (U.S. Pat. No. 6,518,747) teaches the detection of magnetized particles by using Hall effect sensors.
The methods cited above that rely on the use of a GMR sensor, rather than methods such as inductive sensing or Hall effect sensing, will all be adversely affected by the failure of the GMR sensor to maintain a reproducible bias direction for its free layer magnetization. This lack of reproducibility is a result of magnetic hysteresis that occurs whenever the external magnetic fields being detected cause the magnetic moment of the sensor free layer to cycle about its bias direction. In the present use of the GMR sensor to detect the presence of extremely small magnetized particles, the external fields are small. Because of this, methods to fix the bias point of the sensor free layer cannot fix it too strongly as this would limit the ability of the free layer magnetic moment to respond to the very stimuli it is attempting to measure. It is, therefore, necessary to find a way of fixing the free layer bias point while still allowing the magnetic moment sufficient freedom of motion to detect even very small external magnetic fields.